Trophic hormones include thyroid stimulating hormone (TSH), adrenocorticotropic hormone and gonadotropins. Gonadotropins are a kind of glycoprotein hormones that regulate gonadal development of vertebrates and promote the production and secretion of gonadal hormones. For example, the luteotropic hormone (LH) and follicle stimulating hormone (FSH) secreted by the anterior pituitary can function synergistically to stimulate the development of ovarian or testicular germ cells, and the production and secretion of gonadal hormones; and human chorionic gonadotropin (HCG) secreted by human placenta can stimulate the secretion of progesterone from the corpus luteum during pregnancy.
Gonadotropins (e.g., LH, FSH, and HCG) and thyroid stimulating hormones collectively constitute a glycoprotein family, and each of them consists of a α-subunit and a β-subunit, wherein the α-subunits are identical among the hormones, while the β-subunits have characteristics specific for each hormone and therefore determine the biological activities and immuno reactivities of the hormones.
Clinically, FSH is useful in assisted reproductive techniques, such as in vitro fertilization (IVF), gamete intra-fallopian transfer (GIFT) and zygote intra-fallopian transfer (ZIFT), to induce superovulation in patients. In addition, it can also be used for females suffering from an ovulia, including patients with polycystic ovarian syndrome not responsive to clomiphene, as well as for males with pituitary gland hypogonadism. LH is usually used in adjunctive treatments with FSH. hCG is mainly used for promoting ovulation and development of the corpus luteum. For males, it can be used to stimulate Leydig cells to secrete testosterone, to test the functions of Leydig cells, and to promote spermatogenesis when used in combination with agonadotropin in a long-term. TSH is usually used in combination with I131 for inhibiting and ablating the postoperative residual cancer tissue in patients with thyroid cancer.
Generally, long-term treatment is required in order to achieve a therapeutic effect. However, the dosing regimen of daily intramuscular or subcutaneous injection usually results in local reaction and discomfort. Therefore, it would be useful for practical clinical application to develop a long-acting gonadotropin and thyroid stimulating hormone and to reduce the frequency of administration.
For example, FSH can be isolated from the pituitary gland or the urine of postmenopausal women (see, EP322,438), and can also be produced recombinantly(see, U.S. Pat. Nos. 5,639,640, 5,156,957, 4,923,805, 4,840,896, and EP211,894). In order to achieve a therapeutic effect, it takes 8-10 consecutive days, sometimes up to 21 days, to stimulate folliculogenesis in females; and it takes up to 18 months to induce spermatogenesis in males with low gonadotropin level. In recent years, a number of patents and publications reported the development of long-acting FSH, the basic strategy of which is to increase the half-life by increasing glycosylation sites in FSH. One way is to increase the glycosylation modification sites within the FSH molecule. WO01/58493 discloses 77 mutations which can be made in the α-subunit of FSH and 51 mutations that can be made in the β-subunit of FSH. However, this patent does not disclose production or measurement of any FSH α- or β-subunit, into which a glycosylation site(s) is introduced by site-directed mutagenesis. U.S. Pat No. 7,740,862 discloses insertion of the GNFT (SEQ ID NO: 9 or GNRT (SEQ ID NO: 10) sequence between amino acid residue 3 and 4 of the α-subunit of FSH, which results in an in vivo half-life of about 17 h in rats, and an ability to in vitro stimulate CHO cells expressing FSH receptors recombinantly to produce cyclic adenosine monophosphate (cAMP) in a manner comparable to that of the native FSH. Another way is to increase glycosylation modification sites by adding additional sequences to the end of the sequence. One of the relatively successful products developed so far is Elonva, which was marketed by Merck, Germany in 2010. This analog has the comparable biological activity as natural FSH but a longer circulation half-life, which is about 69 h in human. The specific protein sequence is disclosed in U.S. Pat. Nos.5,338,835 and 5,585,345.It contains a modified FSH β-subunit; the C-terminal glutamate of which is extended with the carboxy terminal peptide (CTP) group of hCG. The experimental results of Pieter Verbost, et al. showed that this analog had an in vivo half-life of 17.3 h and 46.9 h in rats and dogs, respectively, which was increased by 1.5-2 olds as compared to that of the recombinant FSH. Signe Perlman, et al. reported a modified FSHα-subunit, with the amino acid sequence “ANITVNITV” (SEQ ID NO: 11) added to the N-terminal. This analogue had an in vivo half-life of 22 h in rats, which was increased by 3-4 folds as compared to that of the recombinant FSH.
IgG immunoglobulin is one of the most abundant proteins in human blood, with a half-life of up to 21 days. Its stability is due to the fact that the Fc fragment of IgG can bind to the neonatal Fc receptor (FcRn), thereby preventing IgG from entering into and being degraded by lysosomes (5-7). Thus, the Fc fragment of IgG is used to link to an active protein to form a fusion protein, thereby increasing the in vivo half-life of the active protein and therefore achieving a long-acting effect. Based on the length of the hinge regions and different amino acid sequences of the Fc fragments, human IgG can be divided into 4 subtypes, among which IgG Fcγ2 has a relatively weak effect of inducing cytotoxicity, and IgG Fcγ1 has the strongest effect. IgG fusion proteins are mostly constructed by linking the N-terminal of the Fc (Hinge-CH2-CH3) fragment or CH (CH1-Hinge-CH2-CH3) fragment of IgG to the C-terminal of an active protein, in order to avoid possible influence of the construction of the fusion protein on the biological activity of the active protein. Furthermore, IgG fusion proteins can be purified efficiently and conveniently by Protein A affinity chromatography. Accordingly, a number of patents have reported that fusion of the Fc fragment of IgG to other proteins can significantly prolong the half-life and increase the in vivo biological activity of the target protein (U.S. Pat. Nos. 5,155,027, 5,428,130, 5,480,981 and 5,808,029).
US patent No. 20050,186,662 discloses that by constructing FSH-Fc fusion protein homodimers and heterodimers, the in vivo half-life in rats was increased to 60 h. As disclosed in the patent, to evaluate the bioactivity of their FSH-Fc fusions, the ovarian weight of rats was measured 72 hours after a single dose of the FSH-Fc fusions or recombinant FSH. The results showed that the ovarian weight in the FSH-Fc fusion protein group (26.9 mg) was increased as compared to the recombinant FSH group (14.3 mg)(the negative control: 12.1 mg). However, due to the sort half-life of the recombinant FSH, daily dosing is usually required in such an activity assay. If administered once every three days according to the protocol, FSH would be substantially metabolized, without any activity. Thus, while ensuring the half-life of the constructed FSH-Fc fusion protein, the activity still remains to be improved.
Therefore, there is a medical need for a product providing full therapeutic effects of gonadotropins or thyroid stimulating hormone, which can be administered less frequently than existing gonadotropin or thyroid stimulating hormone products. In addition, the long acting product can provide a more consistent activity as compared to the activity of gonadotropins or thyroid stimulating hormone achievable by the existing therapies.